DESCRIPTION (provided by applicant: All living cells use signal transduction to detect properties of interest in their environment, create an internal representation of stimuli, and generate an appropriate response to changing conditions. Errors in signal transduction can have serious consequences, such as cell growth without a growth stimulus (i.e. cancer). In both prokaryotes and eukaryotes, information is often encoded as the presence or absence of a phosphoryl group specifically attached to a protein. Understanding the mechanisms and regulation of phosphoryl group transfer among proteins, and the impact of phosphorylation on protein activity, is therefore of broad interest. Because microorganisms constitute the vast majority of life on Earth in terms of both numbers and genetic diversity, microbes are logical subjects in which to seek fundamental biological principles generally applicable to all forms of life. Two-component regulatory systems are widely used for signal transduction by bacteria, archaea, eukaryotic microorganisms, and plants (but not humans). A sensor kinase protein detects stimuli and converts them to phosphoryl groups, which are transferred to a response regulator protein to control responses such as behavior, development, physiology, or virulence. Our long-term goal is to achieve a comprehensive understanding of two-component signal transduction. In this proposal, we will investigate the mechanisms and kinetics by which response regulators switch between phosphorylated (active) and unphosphorylated (inactive) states. In order to synchronize responses with stimuli, the kinetics of signaling biochemistry must match the timescale of the affiliated biological process. Response regulators can self-catalyze phosphoryl group addition and removal. Auxiliary kinases and phosphatases substantially accelerate response regulator autocatalytic reactions to achieve physiologically appropriate signaling speeds, but do not alter the intrinsic reaction mechanisms. Although all response regulators share a conserved structure and catalytic residues, autodephosphorylation rates vary by >40,000x. Aims 1 and 2 focus on identifying factors that control rates of response regulator self dephosphorylation and phosphorylation, and understanding how these elements exert their influence. Our experimental strategy integrates biochemistry, bioinformatics, genetics, and structural biology to alter nonconserved residues in the active sites of various response regulators and determine the functional and structural consequences of doing so. Aims 3 and 4 investigate several specific auxiliary phosphatases (e.g. CheZ, CheX, PhoR) in detail to determine what common mechanistic, regulatory, or structural features may exist among this poorly characterized group of proteins. Antibiotic resistance is a major and increasing threat to human health. This work may impact design of therapeutic agents to attack two-component systems that control virulence or viability of bacterial and fungal pathogens. In addition, the knowledge gained could be used to predict or manipulate the signaling kinetics of two-component systems, or engineer synthetic regulatory circuits with specific timing characteristics.